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Published November 16, 2023 | in press
Journal Article Open

A nanobody-based strategy for rapid and scalable purification of human protein complexes


The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENP^(EuB). We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.

Copyright and License

© Springer Nature Limited 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.


We thank P. Bjorkman for access to her laboratory's cell sorter, as well as the Caltech Flow Cytometry facility. This work was supported by the Heritage Medical Research Institute (R.M.V.), the National Institutes of Health's National Institute Of General Medical Sciences DP2GM137412 (R.M.V.), the Deutsche Forschungsgemeinschaft (T.P.) and the Tianqiao and Chrissy Chen Institute (T.P. and M.H.).


T.A.S., R.M.V. and T.P. conceived and designed this study. T.A.S., G.P.T., M.H., S.W., V.N.N., C.D. and T.P. carried out the experiments and interpreted data. T.A.S., R.M.V. and T.P. wrote the protocol, and all authors provided feedback on its final version.

Data Availability

The lentiviral transfer plasmids and bacterial expression plasmids described in this study are available from Addgene. Addgene IDs of all plasmids are listed in Table 1. Source data are provided with this paper.

Conflict of Interest

R.M.V. and G.P.T. are consultants for Gates Biosciences, and R.M.V. is an equity holder.


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Additional details

November 21, 2023
January 9, 2024